Entomological inferences highlight the risk of Leishmania transmission in the urban area of Porto Velho, Rondônia, Brazil

Entomological investigations were conducted for the first time in urban forest remnants of Porto Velho, state of Rondônia, Brazil, to explore the transmission dynamics of Leishmania. Sand fly collections were carried out at ten sites, encompassing both canopy and ground strata, from October to December 2021. A total of 1,671 sand flies were collected, representing 42 species within 12 genera. Nyssomyia Antunesi (n = 384) and Psychodopygus davisi (n = 111) were the most abundant species. Molecular analyses targeting the V7V8 region (18S gene) unveiled the presence of sequences 100% identical to Leishmania infantum in females of Bichromomyia flaviscutellata (1), Nyssomyia Antunesi complex (6), Nyssomyia umbratilis (1), Nyssomyia sp. (1), Psychodopygus ayrozai (1), Ps. davisi (3), Psychodopygus paraensis (1), and Sciopemyia sordellii (1). Sequences 100% similar to Trypanosoma minasense were found in two samples of the Nyssomyia Antunesi complex, and two samples of Sc. sordellii presented 100% identity to a Trypanosoma sp. strain, previously identified in this same sand fly in Rondônia. Sequencing of Cytb fragment suggested Homo sapiens, Dasypus novemcinctus and Tamandua tetradactyla as the blood source for distinct sand flies. The identification of sequences similar to L. infantum in sand flies collected in urban forest fragments is noteworthy, correlating with the recent local and regional occurrence of autochthonous cases of human visceral leishmaniasis. However, further studies are imperative to ascertain the presence of hosts/reservoirs and evaluate the risk of L. infantum transmission to humans.


Introduction
Sand flies (Diptera, Psychodidae) play a pivotal role as the primary vectors of Leishmania, contributing to the global public health concern caused by leishmaniasis [1].Among the countries significantly affected by both cutaneous (CL) and visceral (VL) forms of leishmaniasis, Brazil stands out, reporting high incidence rates [2].The state of Rondo ˆnia (RO), situated in the northern region, has reported approximately 16,214 CL cases from 2007 to 2022, with sporadic occurrences of VL, totaling eight autochthonous human cases in the same period [3].Notably, the municipalities of Porto Velho and Vilhena exhibit elevated CL-prevalence, with the disease primarily associated with rural and forested areas throughout the state [3].Nevertheless, the epidemiological profile of CL, primary associated with sylvatic areas, has undergone changes, leading to the emergence of autochthonous cases in both rural and periurban areas [4][5][6].
Currently, Rondo ˆnia boasts a documented diversity of approximately 140 sand fly species [7].After Amazonas, thus, Rondo ˆnia has the highest levels of sand fly diversity of all Brazil´s federal states [8].Despite extensive research in rural and sylvatic areas [9,10], the sand fly diversity in urban forest fragments remains unexplored.Consequently, our objective was to: provide the first description of the sand fly fauna in the urban area of Porto Velho; determine the food source in engorged females, and screen for the presence of Trypanosomatidae DNA.

Ethical statements
For sand fly collection, the guidelines of Normative Instruction No. 141, dated December 19, 2006, were followed, which exempts the need for authorization for collection and transportation through the Biodiversity Authorization and Information System (SISBio).Additionally, the project is registered in the National System for Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) with Registration Number AA32B8E.

Study area, collection, and identification of sand flies
Sand fly collections were conducted in October, November, and December 2021 at 10 vegetation sites within the urban area of Porto Velho.Brief descriptions of the sampling locations are provided in (Table 1).Notably, two of the sites, designated as "Parque Natural" and "UNIR," are classified as urban expansion areas according to the Secretaria de Planejamento, Orc ¸amento e Gestão (SEMPOG) of the municipality.The selection of sampling sites was based on the following criteria: (a) the presence of riparian vegetation (fragments of vegetation along riverbanks), (b) the presence of low-order streams, locally called "igarapés", and (c) the presence of human dwellings and/or circulation and domestic animals (Fig 1).
Sand flies were simultaneously collected at the ten points using HP light traps [11] adapted with green LED [10], installed for 12 hours at two different heights, covering distinct strata: one meter above the ground and in the forest canopy at an approximate height of 10 to 12 meters.After each collection, the sand flies were preserved in 92% ethanol and transported to the Laboratory of Entomology at Fiocruz Rondo ˆnia.Male sand flies were directly mounted in Berlese liquid.Female sand flies were initially screened for the presence of residual blood in the abdomen and then dissected to remove the head and the last three tergites for subsequent taxonomic identification [12].The remaining body parts were individually stored in 92% ethanol at -20˚C for molecular procedures.The abbreviations of sand fly genera and subgenera followed [13].

Competing interests:
The authors have declared that no competing interests exist.

Molecular detection of Trypanosomatidae
Prior to DNA extraction, non-engorged female sand flies were organized into groups or individually, each comprising up to twenty specimens from the same month, location, stratum, and species.Whole DNA extraction was performed using the Gentra Puregene Cell and Tissue Kit (Qiagen, Valencia, CA), following the manufacturer's guidelines.To mitigate the risk of cross-contamination, all instruments and workspaces underwent decontamination with DNA-Zap (Ambion Life Technologies, Inc.).Additionally, male sand flies were included as negative control groups throughout all extraction steps.DNA from engorged females was individually processed using the illustra blood genomicPrep Mini Spin Kit (GE Healthcare, Piscataway, NJ), following [14] to prevent cross-contaminations.
The extracted DNA underwent purity assessment using spectrophotometry (NanoDrop), and PCR targeting the cacophony gene in the ISV6 region [15] was utilized to evaluate DNA quality and as an endogenous control for the PCR reactions.
Nested polymerase chain reaction (nested-PCR) was performed targeting the trypanosome barcode, the V7V8 region of the 18S gene [16].In the first stage, a 927 bp fragment was amplified using the primers TRY927F (5'-GAAACAAGAAACACGGGAG-3') and TRY927R (5'-CTACTGGGCAGCTTGGA-3'). In the second stage, a 561-bp internal fragment of V7V8 was amplified using the primers SSU561F (5'-TGGGATAACAAAGGAGCA-3') and SSU561R (5'-CTGAGACTGTAACCTCAAAGC-3') [17].For all PCR assays, a reference strain of Leishmania braziliensis (MHOM/BR/1975/M2903) served as a positive control, and a non-template sample served as a negative control.PCR-positive products were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and subsequently subjected to Sanger sequencing [18].The analysis of electropherograms from both forward and reverse primers was primarily conducted using Finch TV software (Geospiza, Inc., Seattle, USA).Consensus sequences were obtained through BioEdit alignment software and subsequently compared with sequences deposited in the GenBank database using the megaBLAST tool.A cut-off threshold of 98% identity and 100% sequence coverage were applied to confirm the identity of Trypanosomatidae using the V7V8 set of primers.The V7V8 sequences obtained in this study have been deposited in the GenBank database under accession numbers (OR814185-OR814205).

Blood source identification
Engorged female sand flies, characterized by the presence of blood in the midgut, were dissected using sterile needles in PBS 1x.The head and the last three tergites were mounted in Berlese medium for subsequent taxonomic identification [12], and the remaining parts of the body were stored dry at -20˚C until DNA extraction using the illustra blood genomicPrep Mini Spin Kit (GE Healthcare, Piscataway, NJ).Blood meals were identified using the set of primers cytb1 and cytb2, which target a region of approximately 361 bp of the cytochrome B (Cytb) gene [19], the first-choice target for mammal identification [20].DNA from Gallus gallus was used as a positive control, and males of laboratory-reared Lutzomyia longipalpis were used as a negative control to monitor cross-contaminations. PCR-positive products were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and subsequently subjected to Sanger sequencing [17].The electropherogram analysis, construction of consensus sequences, and megaBLAST followed the same protocol described for the molecular detection of Trypanosomatidae.Additionally, a cut-off of 98% identity and 100% sequence coverage were used to ascertain the vertebrate food source of engorged females.The cytb sequences obtained in this study have been deposited in the GenBank database under accession numbers (PP957632-PP957657).

Sand fly fauna
A total of 1,671 specimens were collected, comprising 1,055 specimens in the canopy (63.1%) and 616 on the ground (36.9%).Some females were identified only at the genus level, such as Trichophoromyia spp.and Trichopygomyia spp., while others were identified at the series level, such as Psychodopygus (Chagasi and Guyanensis series), due to morphological damage.Indeed, a total of 1,631 specimens (97.6%) were identified at least at the genus level, comprising 42 species belonging to 12 genera (Table 2).Evandromyia and Psychodopygus were the genera more frequently captured with eight species each, followed by Nyssomyia (six species) and Psathyromyia (five species).However, eight more genera have been recorded in forest fragments within the urban areas: Bichromomyia (one species), Lutzomyia (two species), Micropygomyia (at least one species of Pilosa series), Pintomyia (two species), Sciopemyia (one species), Trichophoromyia (five species), Trichopygomyia (three species), and Viannamyia (two species) (Table 2).The most frequent and abundant species were Nyssomyia antunesi (n = 384) and Psychodopygus davisi (n = 111).
Four samples exhibited similarity to the Trypanosoma genus.Among these, two samples of the Ny.Antunesi complex exhibited 100% similarity to Trypanosoma minasense, previously identified in Madre de Dios region, Peru (Accession number KX932489.1)[21].The remaining two samples, belonging to Sc. sordellii, had 100% similarity to Trypanosoma sp.This trypanosome has previously been identified in the same city (Porto Velho) within the same sand fly [16] (Accession number EU021243.1).All trypanosomatid findings are summarized in Table 3.
Antunesi complex was the most frequently found engorged specimens (N = 7; 24.1%), followed by Ev. piperiformes, Ps. ayrozai and Psychodopygus series Chagasi (N = 3; 10.3% each).A total of three out of 29 samples, belonging to the Nyssomyia Antunesi complex, Ps. claustrei, and Ps.davisi, were Cytb PCR-negative, and the blood source was not assessed.This may be explained by the low amount of DNA or even the advanced digestion process, which is described as a limiting factor in detecting blood sources in sand flies [22].Homo sapiens DNA (N = 20; 77.0%) was found associated with eleven sand fly species, with all sequences presenting 100% similarity to human sequences (Accession number OR608756) identified in a previous study focused on ascertaining the blood source of sand flies [23].Molecular identification of Dasypus novemcinctus (nine-banded armadillo) (N = 3; 11.5%) was observed in two samples of Psychodopygus ayrozai and in one sample of Trichophoromyia sp.One female of Ps. ayrozai presented a mixed food source (H.sapiens/D.novemcinctus).All D. novemcinctus sequences were 100% identical to those from the same vertebrate previously identified in the state of Rondo ˆnia (Accession number MZ447989) [24].Three samples (two belonging to the Nyssomyia Antunesi complex and one of the Chagasi series of the Psychodopygus genus) tested positive for Tamandua tetradactyla (lesser anteater) DNA, and all sequences had 100% similarity to Tamandua tetradactyla sequences (Accession number MW752264) identified in a previous study focused on ascertaining the blood source of Culicoides from Rondo ˆnia [25].Two engorged females also tested positive in V7V8-PCR, and the molecular identification revealed the presence of Trypanosoma minasense DNA in females of the Nyssomyia Antunesi complex that fed on T. tetradactyla and H. sapiens.

Discussion
This study represents the first characterization of the sand fly fauna in urban forest remnants of Porto Velho, Rondo ˆnia, providing insights into species diversity and the potential risk of Leishmania transmission to humans in urban and periurban locations.Although all species collected in this study have been previously documented in Rondo ˆnia [7], this investigation highlights the presence of various vector species in the urban area, some of them presenting major or minor epidemiological significance.
The urban sand fly fauna of Porto Velho comprised at least 42 species, accounting for approximately 31% of the fauna documented in Rondo ˆnia.This may indicate the adaptive potential of several species to urban vegetation areas, particularly in forest fragment sites where domestic and wild mammals are frequently observed.The most dominant species were Nyssomyia antunesi and Psychodopygus davisi, collectively representing approximately 50% of the sand fly fauna.Both species were previously observed in abundance in studies conducted in the periurban area of Porto Velho and various locations across Rondo ˆnia [9,10,26].Regarding Ny. antunesi, in various regions within the Amazonian territory, this species has shown a tendency to establish in human-modified environments [27,28].The precise identification of females of Ny. antunesi remains a challenge.Due to the presence of Ny. urbinattii, which, together with Ny. antunesi and Ny.delsionatali constitutes the 'Nyssomyia Antunesi complex', several females collected in this study were characterized belonging to this complex.
Several species recorded in the urban area of Porto Velho possess epidemiological relevance in the transmission of Leishmania sp., including Br. flaviscutellata, Ny. antunesi, Ny. umbratilis, and several species within the Psychodopygus genus [1].This highlights the potential risk for humans and other host/reservoir species, especially concerning VL.However, notably, fifteen samples showed similarity to L. infantum DNA.The state of Rondo ˆnia reported eight autochthonous human cases of VL between 2007 and 2022, with one documented case in Porto Velho.Therefore, the finding of sand flies carrying L. infantum DNA is of utmost importance and prompts consideration of the possibility of a VL transmission cycle in the urban area of Porto Velho.The occurrence of a sporadic case of VL in Porto Velho, coupled with the identification of PCR-positive sand flies, raises several epidemiological questions that warrant further investigation.Is VL truly sporadic or possibly underestimated in Porto Velho?Despite Brazil's robust disease notification system (Sistema de Informac ¸ão de Agravos de Notificac ¸ão), there is a recognized fragility in the data, considering the potential for underreporting.Another crucial question pertains to the potential host/reservoir.In Brazil, the domestic dog (Canis lupus familiaris) is considered the primary reservoir of L. infantum.Until today, no serological survey has been conducted in Porto Velho to confirm the presence of canine VL.Additionally, no other mammal, whether synanthropic or sylvatic, has been identified as a potential host of L. infantum in the area.Moreover, it is also vital to investigate the potential of the PCR-positive sand fly species in sustaining late-stage infections of L. infantum to determine their role as vectors.Therefore, this study represents a crucial step toward understanding the dynamics of Leishmania transmission in the urban area of Porto Velho.Further studies with diverse focuses are strongly encouraged to deepen our knowledge in this regard.
Beyond Leishmania parasites, this study identified other trypanosomatids carried by female sand flies.The use of the V7V8 region, a trypanosome barcode [16,29,30], as a molecular target allowed the identification of Trypanosoma sp.DNA in females of Sc. sordellii.Here, the parasite exhibited 100% similarity with Trypanosoma sp.deposited in GenBank under accession number EU021243.1.According to the deposit information, this trypanosome was isolated from Sc. sordellii in Rondo ˆnia [16], similar to our observations, where the sand fly species and the study area were the same.Sciopemyia sordellii is known to feed on ectothermic animals, particularly frogs [31], and its role as a Leishmania sp.vector is a subject of debate.Although there is no conclusive evidence regarding its putative capacity to sustain late-stage infections with Leishmania, several studies have identified Sc. sordellii carrying Leishmania DNA [9,[32][33][34].In this study, L. infantum was detected in Sc. sordellii, consistent with previous findings in this sand fly species in the state of Maranhão [34].In periurban areas of Porto Velho, Sc. sordellii has also been found carrying Leishmania naiffi DNA [9].
Trypanosoma minasense was found in two samples of the Ny.Antunesi complex.This parasite belongs to the subgenus Megatrypanum, is a member of the T. irwini clade, and is known to be a simian trypanosome species [35], originally described from black-penciled marmosets (Callithrix penicillata) in Brazil [36].The species has been molecularly detected in several wild primates in South America, especially in saddleback tamarin (Leontocebus weddelli) in southeastern Peru [21], in wild howler monkeys (Alouatta caraya) in northeastern Argentina [37], and in a South American red-handed tamarin (Saguinus midas) [38].The sequence of V7V8 The sequence of the V7V8 fragment showed that T. minasense from Rondo ˆnia was 100% identical to Peruvian isolates (Accession number KX932489.1)[21], and the geographical proximity between these areas may explain the similarity.
The detection of trypanosomes in sand flies are often by-product of epidemiological studies on leishmaniasis.Various sand fly species have been reported infected with trypanosomes; however, few parasites have been isolated and well-characterized.The lack of knowledge regarding the food habits of sand fly species hampers the association with potential hosts and consequently, trypanosome species.Sand flies belonging to the genera Evandromyia, Lutzomyia, Psathyromyia, and Sciopemyia, captured in soil and tree trunks, have been reported to be naturally infected with anuran Trypanosoma DNA in the state of Rondo ˆnia [16].Circumstantial evidence has suggested that neotropical sand flies may be vectors of rodent and bat trypanosomes of the subgenus Megatrypanum.Trypanosoma freitasi has been found naturally infecting Psychodopygus claustrei from Amazonas, Brazil [39], and triatomine bugs appear to be refractory to this parasite [40].
Most of the engorged females belonged to the Nyssomyia Antunesi complex and were found in both canopy and ground strata, similarly to reported in the state of Para ´, where this species appears to switch between those strata, probably to feed on different vertebrates, indicating its eclectic feeding habit [41].Five out of seven (71%) of engorged females of Ny.Antunesi complex fed on H. sapiens, and the remaining fed on T. tetradactyla.Notably, the two females of Ny.Antunesi complex positive for T. minasense fed on T. tetradactyla and H. sapiens, both collected in the canopy.The remaining species Ev. georgii, Ev. piperirformis, Ev. walkeri, Ny. fraihai, Ny. richardwardi, Ny. umbratilis, Ps. ayrozai, Ps. carrerai, Ps. davisi, and Psychodopygus series Chagasi fed on H. sapiens.Several of these species have had their food habits identified for the first time, and further studies are necessary to ascertain their capacity to feed on other vertebrates.Psychodopygus davisi and Ps.ayrozai have been previously reported in studies in the state of Rondo ˆnia feeding on humans [42], and the latter species was detected with mixed feeding on D. novemcictus and H. sapiens, indicating the opportunistic behavior of the species.
This study provided valuable information on the diversity of sand fly species and potential risks of Leishmania transmission to humans and other hosts/reservoir in urban areas of Porto Velho.The report of L. infantum DNA in sand flies indicates the need to carry out a survey to identify potential hosts//reservoirs in the study area.The high abundance of Ny. antunesi and Ps.davisi suggests an adaptation of these species to human-modified environments.The presence of these species in urban areas is particularly relevant due to their potential as vectors of Leishmania sp., highlighting the risk to human and animal health.Furthermore, the detection of T. minasense DNA in females of the Ny.Antunesi complex emphasizes the complexity of parasitic interactions in this ecosystem.

Fig 1 .
Fig 1. Illustrative images of some localities where traps were installed, depicting small forest fragments in urban environments and the presence of animals such as chickens.Images under a CC BY license, with permission from Michelli Santos da Silva, copyright 2023.https://doi.org/10.1371/journal.pone.0309168.g001

Table 1 . Geographical coordinates and brief descriptions of the sampling sites in Porto Velho, RO, Brazil. Sampling sites Geographical coordinates Description
2 size fragment of forest in a neighborhood, scout headquarters with protected area for a water spring and used for recreational purposes, presence of domestic and wild animals.São João Bosco 8˚44'35.54"S635 4'5.73"W 0.1km 2 size fragment of forest in a neighborhood, wild and domestic animals, area with solid waste disposal.https://doi.org/10.1371/journal.pone.0309168.t001

Table 3 . Molecular detection of Trypanosomatidae in females collected in October, November, and December 2021 in forest fragments in the urban area of Porto Velho, Rondo ˆnia, Brazil. Species Total of individual / Pool samples Positivity for Leishmania/Trypanosoma
* Two Samples from Ny. Antunesi complex positive for T. minasense DNA.** Two samples from Sc. sordellii positive for Trypanosoma sp.DNA.https://doi.org/10.1371/journal.pone.0309168.t003

Table 4 . Engorged females collected in canopy and ground strata in forest fragments in the urban area of Porto Velho, Rondo ˆnia, Brazil.
The two females of Ny. antunesi complex positive for T. minasense fed on Tamandua tetradactyla and Homo sapiens.One female of Ps. ayrozai presented mixed food source (H.sapiens/D.novemcintus).
a b